Choline is a dietary nutrient essential for the structural integrity and signaling functions of cell membranes;it is the major source of methyl-groups in the diet, and it directly affects cholinergic neurotransmission, transmembrane signaling and lipid transport/metabolism. The major premise for this proposal is that humans require a dietary source of choline and that this requirement has significant individual variation. We suggest that estrogen and common genetic polymorphisms modulate this dietary requirement for choline. During the last 4 years of funding we established that 77% of men and 80% of postmenopausal women, when deprived of dietary choline, developed fatty liver or muscle damage. Only 43% of premenopausal women developed signs of organ dysfunction associated with choline deficiency. Some men developed such signs of choline deficiency when ingesting the presumed Adequate Intake for choline (550 mg/day). We observed that the PEMT promoter is estrogen responsive, and we hypothesize that estrogen status influences the dietary requirement for choline. We identified common genetic variations that appear to increase the likelihood that a human will become choline deficient when fed a low choline diet;postmenopausal women with a common single nucleotide polymorphism (snp) in the promoter region of the gene responsible for endogenous biosynthesis of choline (phosphatidylethanolamine N-methyltransferase, PEMT;rs12325817) had a odds ratio (OR) compared to wild type of 42 for developing signs of organ dysfunction when we removed choline from their diets (p=0.03);premenopausal women with a G78T snp in choline dehydrogenase had an OR of 20 (p=0.04) and premenopausal women with a common snp in methylene tetrahydrofolate dehydrogenase (MTHFD1-1958A) had an OR of >85 (p=.0001) for developing signs of choline deficiency. We identified another snp in choline dehydrogenase A40 that was associated with a decreased susceptibility to developing signs of choline deficiency in all subjects (OR = 0.2;p = 0.03). These snps are common in humans, for example, for the PEMT promoter snp 18% of our subjects were homozygous for the snp, 56% were heterozygous, and 26% were wild type. Therefore, it is important that we have a better understanding of how these genetic polymorphisms influence the dietary requirement for choline. We propose studies that will extend our observations during the last funding period and that will refine our understanding of factors that influence dietary requirements for choline in humans. Experiments are proposed that will determine whether postmenopausal women treated with estrogen have a lower choline requirement, identify functionally important SNPs, determine prevalence of these in the population and examine dietary choline requirements in humans with these SNPs.